The present invention relates to media for culturing human cells comprising human serum and a method for culturing human cells using the same.
Typically, since it is difficult to regenerate cartilages, it is thus very difficult to treat diseases which occur therein such as arthritis.
To treat such a disease, a method was developed for culturing and proliferating patient""s own chondrocytes in vitro, and grafting them to a diseased part thereafter. To accomplish such self-grafting, chondrocytes as many as possible are required and the peculiar expression-types of chondrocytes such as second-type collagen proteoglycan, etc. must also be maintained.
A present laboratorial method for culturing chondrocytes used chiefly for cell grafting is to perform monolayered-culture of chondrocytes in appropriate media, then to proliferate them, to three-dimensionally culture them in alginate beads and to reconstruct the peculiar expression-types of the chondrocytes thereafter.
Since proliferation speed of chondrocytes, however, is very slow, long time is consumed to obtain a proper number of chondrocytes even though they are monolayer-cultured. In this process chondrocytes are dedifferentiated and three-dimensional culture is applied to reconstruct the expression types of the chondrocytes. But the problem is that the number of chondrocytes does not increase in this process.
It is an object of the invention to provide media that can be used for culturing human cells including chondrocytes without generating conventional problems such as proliferation speed delay and loss of an expression types.
It is also another object of the invention to provide a method for culturing human cells using the aforementioned media.
The present invention is characterized in that the media used for culturing human cells comprise human serum, wherein the method comprises culturing human cells in the aforementioned media.
In a preferred embodiment, the media used for culturing the human cells comprise human serum of 1 to 50%(v/v).
In a preferred embodiment, the human, serum comprised in the media is originated from the blood of AB-type.
In a preferred embodiment, human cells cultured in the media are chondrocytes.
In a preferred embodiment, the media used for culturing the human cells comprise at least one selected from the group of including insulin, transferrin, selenium. T3 hormone and a mixture thereof.
In a preferred embodiment of the invention, the media used for culturing the human cells comprise at least one selected from the group of including a transforming growth factor (TGF-xcex2), an insulin-like growth factor (IGF), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a keratinocyte growth factor (KGF), an epidermic growth factor (EGF) and a mixture thereof.
In a preferred embodiment, for the media comprising the human serum and a method for culturing human cells cultured in the media, the serum comprised in the media and the cells cultured therein are originated from the same person.
The media for culturing human cells comprising human serum and a method for culturing human cells using the same in accordance with this invention are described in detail hereinafter.
It is preferred to derive the human serum used particularly in this invention from the blood of a patient who has given the cultured cells, but it may be obtained from a blood bank, and so on. Human serum from all blood types of O, A, B and AB can be used, but that from AB-type is preferred in particular. Human serum increases the proliferation speed of human cells and allows their expression type stably manifested. The amount of human serum of 1 to 50% (v/v), preferably 5 to 20% (v/v), may be added and used in the media for culturing human cells.
For the media for culturing human cells in which human serum may be added, all types of media generally used may be used. Examples of representative media include DMEM (Gibco BRL), Han""s F-12 media (Gibco BRL), and so on.
Also, the media of this invention may further comprise at least one selected from the group including human-insulin, human-transferrin, sodium selenite, T3 hormone (3,3,5-triiodo-L-thryonine) and a mixture thereof. The amount of human-insulin and transferrin may be 1 mg/l through 15 mg/l respectively, and that of sodium selenite and T3 hormone may be 1 xcexcg/l through 15 mg/l respectively. The media may further comprise a transforming growth factor-xcex2 (TGF-xcex2), an insulin-like growth factor (IGF) a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a keratinocyte growth factor (KGF), a epidermal growth factor (EGF) and a mixture thereof. And the used amount thereof may be 1 xcexcg/l through 10 xcexcg/l.
Human cells that may be cultured in the media may be chondrocytes, liver cells, bone cells, nerve cells, myoblasts, and so on, but chondrocytes are most preferred. Human cells may be obtained from the cells separated by processing human tissues with appropriate ferments. For example, chondrocytes are obtained from the cells separated by processing chondrocyte tissues on both ends of a bone with hyaluronidase, trypsin, collagenase, etc. In particular, it is preferred to use human cells which are the patient""s own cells who needs cell grafting.
Culturing of human cells may be performed according to any one of the known typical methods. A representative example is to perform primary cultivation of the chondrocytes for 5 to 28 days at the temperature of 35 to 39xc2x0 C. with aeration of carbon dioxide of 5 to 15% in the media, and then three-dimensional cultivation of the chondrocytes, dedifferentiated in the process, for 3 to 14 days at 35 to 39xc2x0 C. under carbon dioxide of 5 to 15% after inserting them into alginate beads.
The advantages of the method in accordance with the invention are that the culturing period can be shortened and the expression types of the cultivated cells can be stably manifested. The human cells obtained by the method in accordance with the invention may be used advantageously for cell or tissue grafting and has no considerable side effects along with the grafting.